β-Lactoglobulin (βLG) is a basic element of globular carrier protein, which is the major protein in the whey of ruminant milk and is of main interest in the dairy industry. In the present study, the simultaneous effects of both of the important anticancer drugs of 5-fluorouracil (5-FU) and oxali-palladium, on the structure of βLG were investigated using different spectroscopic methods of fluorescence and circular dichroism (CD) in combination with a molecular docking at two temperatures of 25 and 37 °C. The resulted data from intrinsic fluorescence spectra of protein indicated that 5-FU and oxalli-palladium can quench the fluorescence intensity of βLG in dose-dependent manner via static mechanism of fluorescence quenching. Analysis of fluorescence quenching data in agreement with theoretical results have represented that there are I binding sites on βLG for binding of oxali-palladium and also II binding sites for 5-FU, at both temperatures of 25 and 37 °C. Also, competitive binding results showed that the number of binding sites on protein for each of the drug when the protein incubated with one of the drug did not show any changes. The values of thermodynamic parameters of ΔH°, ΔS° and ΔG° illustrate that van der Waals and hydrogen-bond interactions have the main role in the binding of oxali-palladium and 5-FU to βLG, respectively. The analysis of circular dichroism spectra indicated reduction in stability of the protein and alteration in the secondary structure of protein with reduction of α-helical structure and increasing of β-sheet structure in the presence of increasing concentration of oxali-palladium and 5-FU. Also, the transition temperature (Tm) value of βLG indicated the significant decreasing in the presence of 5-FU and oxali-palladium. As a result, it can be concluded that both of the chemotherapeutic drugs of oxali-palladium and 5-FU can bind to independent binding sites on carrier protein of βLG, which can be used in design and simultaneous delivery of both drugs.